Download Protocol for staining cells with organelle

Staining cells and Microscopy
Lenzmeier Research Laboratory
Protocol for staining cells with organelle-specific agents:
1. Preparing cultures for staining:
12-48 hours prior to doing your experiment you should split cells fresh onto containers that
can be used for imaging using the confocal microscope—grow cells in 6 well plates or the small
tissue culture petri dishes in 3 ml of complete medium per well/dish. Ideally your cells would be
50-75% confluent when carrying out the staining experiment.
2. Stains available:
The stains below are available at BVU. Please note, you can stain with one red and one green
fluorophore simultaneously in the same dish but should not use multiple red or multiple green
fluorophores in the same dish.
Structure
Color Stain
Mitochondria Red Mitotracker Deep Red FM
Lysosomes
Red Lysotracker Deep Red FM
Cell Surface
Green WGA-Alexafluor-488
Cell Surface
Red WGA-Alexafluor-633
Nucleus (NAs) Green Syto9
Nucleus (NAs) Red NucRed Live-647
[Stock]
200 M
10 M
1 mg/ml
1 mg/ml
10 M
N/A
Solvent Aliquot amts
DMSO
60 l
DMSO
100 l
PBS
125 l
PBS
125 l
DMSO
100 l
PBS
2.5 ml bottles
Location
cold room
cold room
-20 freezer
-20 freezer
cold room
cold room
How to make stock solution
1 tube in 460 ul DMSO
50 l in 5 ml DMSO
5 mg bottle in 5 ml PBS
5 mg bottle in 5 ml PBS
10 l from tube in 5 ml DMSO
Use tubes from manufacturer
3. Staining the cells:
At the time you are going to stain the cells be sure to:
 Set out your PBS in the hood so it is at room temp when you do the washes immediately
prior to microscopy.
 Turn on the confocal microscope and let it warm up for 30 minutes:
o Follow the stepwise startup procedures for turning on the lasers and microscope.
o Log into the computer (be sure to not let it do the drive scans) using the username
“Administrator” with a password of “fluoview.”
o Log into the imaging software use the username “Lenzmeier” with a password of
“Lenzmeier”.

Use the table on the
right as a guide your
staining and following
the procedures on the
next page.
Color Stain
Mitochondria
Red Mitotracker Deep Red FM
6 l
30 min @ 37oC
Lysosomes
Red Lysotracker Deep Red FM
15 l
30 min @ 37oC
Cell Surface
Green WGA-Alexafluor-488
20 l
30 min @ 37oC
Cell Surface
Red WGA-Alexafluor-633
20 l
30 min @ 37oC
15 l
30 min @ 37oC
Nucleus (NAs) Green Syto9
Nucleus (NAs)
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l per 3ml dish stain time/temp
Structure
Red NucRed Live-647
150 l (3 drops) 30 min @ 37oC
Prepare a mix of stain/medium. For example, if you are staining 6 petri dishes with
mitotracker deep red FM and WGA alexafluor 488:
o Place 18 ml of medium (3 ml per dish being stained) into a sterile 50 ml tube
o Add 36 l of mitotracker deep red FM (6 l per dish being stained; see table
above)
o Add 120 l of WGA-Alexafluor-488 (20 l per dish being stained; see table
above)
Mix the stain/medium tube well by inversion several times.
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Staining cells and Microscopy
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Lenzmeier Research Laboratory
Remove the original medium from the dishes being stained and add 3 ml of stain/medium
into each dish.
Incubate dishes 30 minutes at 37oC.
Remove stain/medium and discard into waste
Wash each dish gently with 3 ml of PBS and then remove PBS and discard into waste
Add 3 ml of fresh PBS to each dish and proceed to the confocal microscope for imaging.
4. Obtaining images of the cells using the confocal microscope:
A. Initial focusing with the transillumininator lamp:
 Turn on the transilluminator lamp using the button in the upper left corner of the Image
Acquisition Control panel on the computer screen
 Push in the deflector on the microscope to the eye setting
 Set the filter wheel on the microscope to #6 (dict)
 Open the shutter by sliding the knob on the microscope from to O
 Place your sample on the microscope platform and use the 10X objective
 Focus using the coarse focus green buttons on the microscope until your cells are in view
 Use the fine focus adjustment knob until you feel your cells are focused appropriately.
 Turn off the transilluminator lamp by clicking the button in the upper left corner of the
Image Acquisition Control Panel on the computer screen
 Pull out the deflector on the microscope to the camera setting
 Set the filter wheel on the microscope to #1 (LSM)
 Leave the shutter open on the setting open on the microscope (setting of O)
B. Obtaining fluorescent images
In the acquisition settings panel:
 Change the “Aspect Ratio” to either 512X512 or 320X320
 Make sure the correct objective has been chosen (usually UPLAPO 10X NA:0.40)
In the Image acquisition control panel:
 Make sure the box for “Kahlman” filter and “Sequential” are not checked in the bottom
of this panel.
 Select the lamp from the dye list depending on your fluorophor by clicking on the green
circle button (upper left corner third button down). You will need to click “all clear” and
then scroll down in the dye list below, find your dye(s), double click the dye(s) you want,
and then click the “Apply” button. The table below contains a list of what settings to use
for each stain:
Structure
Color Stain
Mitochondria Red Mitotracker Deep Red FM
Lysosomes
Red Lysotracker Deep Red FM
Cell Surface
Green WGA-Alexafluor-488
Cell Surface
Red WGA-Alexafluor-633
Nucleus (NAs) Green Syto9
Nucleus (NAs) Red NucRed Live-647
Exitation l
644 nm
644 nm
495 nm
632 nm
485 nm
638 nm
Emission l
665 nm
668 nm
519 nm
647 nm
498 nm
686 nm
Microscope filter setting
Alexafluor 647 dye
Alexafluor 647 dye
Alexafluor 488 dye
Alexafluor 647 dye
Alexafluor 488 dye
Alexafluor 647 dye
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Staining cells and Microscopy
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Once you’ve selected the dye(s) you want, close the dye list panel.
Click on “XY repeat” to start viewing a live fluorescent image of your cells. You may
not see anything…you will need to consider the following issues in obtaining your
images:
o You might need to focus using the fine focus on the microscope
o You might need to alter the laser intensity by clicking changing the value for HV
for that dye
o You might need to alter the “gain” setting. Increasing this number makes the
brightness (fluorescence) brighter but also increases the background noise.
o You might need to alter the “offset” setting. Increasing this number makes the
darks darker (decreases background but takes away from the signal.
o You might need to alter all of the above settings to get the best photo.
o You need to pay attention to bleaching of your fluorophors. Repeated excitation
leads to a loss of fluorescence. Move quickly to focus to avoid photobleaching.
Once you have an image you like, click the “repeat stop” button (the second button to the
right of XY repeat.
Record the settings you used in your lab notebook so this can be repeated at future times.
In the acquisition settings panel:
 Set the “Aspect Ratio” to the desired resolution. Typically this will be 800X800 or
1024x1024.
In the Image acquisition control panel:
 Click on the Kahlman filter box toward the bottom of the panel. You may wish to get an
image with this to improve crispness, but it adds to bleaching because it takes longer to
obtain the image. Try it and see if you want to use this for your images.
 Click on the “XY” button toward the top of the panel to obtain your image. Adjust
settings accordingly for future images.
Note: If wanting to simultaneously examine multiple fluorophors, optimize the settings for HV,
gain and offset for each fluorophor and write them down. Then choose all the fluorophor
excitation/emissions from the dye list and make sure the optimal HV, gain and offset settings
correspond to each dye. When you do your image acquisition you will want to click on the
“sequential” box at the bottom of the panel and then obtain your image using “XY.”
C. Saving fluorescent images
 Go under “file” and save your image to your folder. Save it immediately as an .obi file.
You will not be able to open this type of file on your computer but will want to save it so
you can access all the quantitative information embedded in the file.
 To obtain images that you can open and process/quantitate on your computer, go under
the file tab and “export as tif.”
D. Transferring fluorescent images to your computer
 Use the usb flash drive to transfer the .tif files of your images from the hard drive to your
computer.
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Staining cells and Microscopy
Lenzmeier Research Laboratory
5. Turning off the computer and microscope components:
 Close out of the software and transfer your files.
 Shut down the computer
 On the microscope:
o Push in the deflector on the microscope to the eye setting
o Set the filter wheel on the microscope to #6 (dict)
o Close the shutter by sliding the knob on the microscope from O to
o Place the microscope dust cover back over the microscope
 Follow the stepwise instructions next to the microscope for how to turn off the
microscope components. Be sure to wait until the laser cooling fan shuts off before
flipping its switch to off.
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